Protein kinases are allosteric enzymes involved in cell signaling pathways (including maturation, differentiation, and metabolism) and many pathological diseases (cancer, diabetes, rheumatoid arthritis, cardiomyopathy, and others). In this proposal, we will focus on the study of allosteric binding processes of protein kinase A (PKA), a prototypical enzyme for the kinase superfamily. Although PKA has been studied for several decades, the molecular mechanisms for substrate recognition, as well as endogenous and exogenous inhibitors are still elusive. There is compelling evidence that these phenomena are mediated by the enzyme's internal dynamics. For PKA, ligand binding (nucleotides, substrates, and inhibitors) modulates the dynamic state of the enzyme, with direct repercussions on the catalytic turnover. Here, we will use a combination of high-resolution techniques (X-ray, NMR, and MD computer simulations) as well as biophysical approaches (thermocalorimetry, kinetic assays, and H/D coupled to mass spectrometry) to trace the allosteric cooperativity in PKA. In AIM 1, we will dissect the allosteric pathways using dysfunctional mutants. In the AIM 2, we will study the dynamic activation and deactivation of the kinase by nucleotides and ATP-competitive inhibitors. Finally, in AIM 3, we will focus on the endogenous inhibition of PKI (protein kinase inhibitor) and its effects on the enzyme's internal dynamics. Understanding how allosteric signals propagate and trigger binding cooperativity will help in designing new strategies to control (by inhibiting or tuning) kinase activity for innovative therapies to treat disease.